薑黃素使乳癌細胞產生細胞週期停滯和走向細胞凋亡

Abstract

在女性的罹癌率中，乳癌是最常見的癌症，而其死亡率是女性癌症中的第二位。乳癌可以簡單的被分為三類，分別是：有表現雌激素接受器 (ER-positive)，或者是在HER2蛋白質方面有過度表現，最後一種是既沒有表現雌激素接受器也沒有HER2蛋白質過度表現的現象。現今已經有了可以分別針對雌激素接受器或是HER2蛋白過度表現的乳癌的治療方式，稱做標靶治療 (Target therapy)，像是tamoxifen和Transtuzmab (herceptin)。然而在雌激素接受器和HER2兩者都呈現陰性的乳癌方面，則是沒有任何專一性的藥物可用於治療。現在第二型S週期激酶相關蛋白 (S phase kinase-associated protein 2，Skp2)被普遍認為是一種原致癌基因 (proto-oncoprotein)，在許多的研究中證明，第二型S周期激酶相關蛋白 (Skp2)會藉由泛素 (ubiquitin)參與在p27kip1蛋白降解的過程中，使p27蛋白的表現量減少。 臨床研究上發現，在雌激素接受器陰性和HER2陰性的乳癌中，大部分都有Skp2過度表現的現象，約佔了61-67%，而在雌激素接受器陽性的乳癌中，有15-23%的病患有Skp2過度表現的情形，也佔了相當高的比例。此外，在近年來的研究中發現，Skp2過度表現在抗雌激素治療無效的過程中扮演了非常重要的角色。然而，目前還沒有專一性的標靶藥物可用於抑制Skp2的表現。 薑黃素 (curcumin)已經被廣泛的報導出具有抗發炎、抗氧化、抑制生長和抗血管新生的功能。除此之外，有很多的研究也指出，在許多種類的癌症中，薑黃素可以有效的去抑制癌細胞的生長。根據這些研究的成果顯示薑黃素可以當成癌症化學預防及化學治療的藥物。 在本文的研究中，我們使用Skp2和HER2都過度表現的乳癌細胞MDA-MB-231/Her2做實驗，去觀察薑黃素對於這株乳癌細胞在細胞增殖、細胞週期調控、促進細胞凋亡，以及一些相關分子的改變會造成怎樣的影響。我們藉由流式細胞儀去分析薑黃素在細胞週期以及細胞凋亡方面所造成的改變。根據其結果，發現到在加入薑黃素之後，隨著劑量不同會有不一樣的影響。在30μM時，會使細胞停留在細胞週期中的G1階段，而在加入50μM的薑黃素後，則是可以看到有效的使細胞走向細胞凋亡。接著我們進一步的去觀察薑黃素對那些調控細胞週期的蛋白質表現量所造成的改變。我們發現了薑黃素會增加細胞中p27Kip1蛋白，同時會減少Skp2、cyclin E和CDK激酶蛋白的表現量，而且這樣的改變會隨著時間拉長和劑量的增加越趨於明顯。這樣的結果也指出Skp2 和p27蛋白質有參與在薑黃素抑制細胞增殖的過程中，並且扮演著重要的角色。另外，隨著加入的薑黃素劑量的增加，走向細胞凋亡的細胞會越來越多，這樣的改變跟活化的caspase 3和PARP蛋白量增加有關。值得注意的是，在本篇研究中，我們透過反轉錄聚合酶鏈式反應 (RT-PCR)發現了薑黃素可以藉由減少Skp2 mRNA的表現量進而有效的去降低Skp2蛋白質的表現量。 綜合這些結果我們得到了結論，不同劑量的薑黃素在乳癌細胞中，可藉由調控與細胞週期相關分子的蛋白表現量和活化的caspase 3和PARP蛋白表現量，分別引發細胞週期停滯和細胞凋亡。薑黃素也可直接藉由抑制mRNA的表現量的方式去降低Skp2蛋白質的表現。根據我們的研究成果，我們認為薑黃素或許可以有效的被利用於乳癌的治療，特別是在具有Skp2過度表現的乳癌中。

In women, breast cancer is the most common cancer, and the secondary occasion of cancer-related deaths. It can simply divide breast cancer into estrogen receptor-positive (ER-positive) and HER2-overexpression or both ER and HER2 negative. Target therapies, which are direct against estrogen receptor and HER2 amplification are existence. Nevertheless, there is no specific therapy which has been identified in tumors that are both ER and HER2 negative. The S-phase kinase-associated protein Skp2 is presumed a proto-oncogene. It has been reported involving in the ubiquitin mediated degradation of p27. In clinical studies, Skp2 overexpression is in the majority in ER/HER2-negative tumor, about 61-67%. And it is also expressed in 15-23% of the ER-positive tumor. In present studies, Skp2 may play an important role in antiestrogens resistance. However, there is no drug which can specific target to Skp2 right now. Curcumin has been reported that it has the functions of anti-inflammatory, antioxidant, antiproliferative, and antiangiogenic. Moreover, there are numerous studies which establish that curcumin can retard proliferation of many kinds of cancer. These data indicated that curcumin may be used as a possible chemoprotective or chemotherapeutic agent. Here, we measured the effect of curcumin on human breast cancer cell (MDA-MB-231/HER2) proliferation, cell cycle regulation and apoptosis induction and associated molecular alterations. Flow cytometric analysis was used to analyze the effects of curcumin on cell cycle and quantify apoptosis. Curcumin treatment of these cells resulted in a G1 arrest at lower dosage (30 μM), and a significant apoptosis at higher dosage (50 μM). We examined the effects of curcumin on cell cycle regulatory protein expression. We found that curcumin increased p27Kip1 and decreased Skp2, Cyclin E, CDK kinases in a time- and dose-dependent manner, suggesting that p27Kip1 and Skp2 may be involved in the growth inhibition by curcumin in MDA-MB-231/HER2 cells. Furthermore, curcumin showed a dose-dependent apoptotic death in MDA-MB-231/HER2 cells which was related to cleaved forms of PARP and caspase 3. Moreover, curcumin repressed the mRNA level of Skp2 which was detected by reverse transcription polymerase chain reaction (RT-PCR). Together, these results suggest that curcumin can trigger cell cycle arrest and apoptosis at different dosages by regulating cell cycle associated molecular expression and activating PARP and cleaved caspase 3. Besides, the result of reverse transcription polymerase chain reaction (RT-PCR) demonstrated that curcumin reduced the expression level of Skp2 protein on the transcription level. In addition, our findings indicate that curcumin is of potential value for the chemoprevention of breast cancer, especially in breast cancer with Skp2 overexpression.